Gene expression alterations of human liver cancer cells following borax exposure.

Borax is a boron compound that is becoming widely recognized for its biological effects, including lipid peroxidation, cytotoxicity, genotoxicity, antioxidant activity and potential therapeutic benefits. However, it remains unknown whether exposure of human liver cancer (HepG2) cells to borax affects the gene expression of these cells. HepG2 cells were treated with 4 mM borax for either 2 or 24 h. Gene expression analysis was performed using Affymetrix GeneChip Human Gene 2.0 ST Arrays, which was followed by gene ontology analysis and pathway analysis. The clustering result was validated using reverse transcription­quantitative polymerase chain reaction. A cell proliferation assay was performed using Celigo Image Cytometer Instrumentation. Following this, 2­ or 24­h exposure to borax significantly altered the expression level of a number of genes in HepG2 cells, specifically 530 genes (384 upregulated and 146 downregulated) or 1,763 genes (1,044 upregulated and 719 downregulated) compared with the control group, respectively (≥2­fold; P<0.05). Twenty downregulated genes were abundantly expressed in HepG2 cells under normal conditions. Furthermore, the growth of HepG2 cells was inhibited through the downregulation of PRUNE1, NBPF1, PPcaspase­1, UPF2 and MBTPS1 (≥1.5­fold, P<0.05). The dysregulated genes potentially serve important roles in various biological processes, including the inflammation response, stress response, cellular growth, proliferation, apoptosis and tumorigenesis/oncolysis.

Overexpression and correlation of HIF-2α, VEGFA and EphA2 in residual hepatocellular carcinoma following high-intensity focused ultrasound treatment: Implications for tumor recurrence and progression.

Rapid growth of residual tumors can occur as a result of their recurrence and progression. The present study aimed to investigate the expression of hypoxia inducible factor-2 subunit α (HIF-2α), vascular endothelial growth factor A (VEGFA), erythropoietin-producing hepatocellular A2 (EphA2) and angiogenesis in residual hepatocellular carcinoma (HCC), following treatment with high-intensity focused ultrasound (HIFU) ablation, in order to investigate the association between protein expression and tumor recurrence and growth. Athymic BALB/c (nu/nu) mice were subcutaneously inoculated with the HCC cell line HepG2, in order to create xenograft tumors. Approximately 30 days post-inoculation, eight mice were treated with HIFU, whereas eight mice received no treatment and acted as the control group. Residual tumor tissues were obtained from the experimental groups after one month. Levels of HIF-2α, VEGFA, EphA2 and cluster of differentiation 31 (CD31) expression was measured by immunohistochemical staining. CD31-positive vascular endothelial cells were counted to calculate microvascular density (MVD), and western blot analysis was performed to determine levels of HIF-2α, VEGFA, and EphA2 protein. It was found that the expression levels of HIF-2α, VEGFA, EphA2, and MVD proteins in residual HCC tissues were significantly higher than in the control group tissues (P<0.05). Tumor MVD was strongly correlated with VEGFA (R=0.957, P<0.01) and EphA2 (R=0.993, P<0.01) protein expression levels. Furthermore, there was a significant positive correlation between HIF-2α and EphA2 expression (R=0.991, P<0.01). The correlation between VEGFA and EphA2 expression was also positive (R=0.985, P<0.01). These data suggest that overexpression of HIF-2α, VEGFA and EphA2 is related to angiogenesis in residual HCC following HIFU ablation, potentially via their association with key mediators of recurrence.

Erythropoietin combined with granulocyte colony­stimulating factor enhances MMP-2 expression in mesenchymal stem cells and promotes cell migration.

Mesenchymal stem cells (MSCs) are promising clinical tools, but the molecular mechanisms that regulate the mobilization and homing of MSCs and cause invasion through extracellular matrix (ECM) barriers are unknown. Matrix metalloproteinase (MMP) degrades the ECM and promotes cell migration. In this study, we investigated MMP expression and cell migration after treatment with erythropoietin (EPO) and granulocyte colony-stimulating factor (G-CSF). Specifically, we studied whether EPO combined with G-CSF enhanced MMP expression and increased the in vitro motility of MSCs. Real-time PCR was used to detect the mRNA of MMPs and tissue inhibitors of metalloproteinase. Cell migration was evaluated by transwell and wound healing assays. Western blotting was used to detect changes in ERK1/2 protein levels. The results showed that EPO combined with G-CSF enhanced MMP-2 expression in MSCs, promoted MSC motility and activated the ERK1/2 signaling pathway. Thus, a combination treatment of EPO with G-CSF promoted cell migration by stimulating MMP-2 expression in MSCs and this appeared to be related to the ERK1/2 signaling pathway.

Increased expression of IL-6 mRNA in hepatocellular carcinoma cell lines correlates with biological characteristics.

BACKGROUND AND AIMS: IL-6 has been implicated in both virus-associated and diethylnitrosamine-induced hepatocellular carcinomas (HCCs). Generally it is produced by immune cells such as Kupffer cells in the liver. To understand mechanisms by which IL-6 might participate in the genesis of HCCs, the production of IL-6 by cell lines under different conditions was examined to determine inducing factors. METHODS: Expression of IL-6 mRNA in both hepatoma cell lines and a normal liver cell line L-02 was measured by quantitative RT-PCR. Biological molecules including liposome, dsRNA and cell debris were used to stimulate IL-6 mRNA expression in HepG2 cells and inhibition was effected by RNAi. Proliferation was assessed by MTT and clone formation and migration was determined by scratch assay. RESULTS: All of the HCC cell lines observed expressed IL-6 mRNA, including HepG2, Bel-7402(7402), MHCC-97H and SMMC-7721.Normal liver cell line L-02 also expressed IL-6 mRNA. SiRNA to IL-6 specifically knockdowned IL-6 mRNA expression in HepG2, and liposome, dsRNA and cell debris increased it. Both proliferation and migration of HepG2 cells were related to the level of IL-6 HepG2 expressed. CONCLUSION: Both normal liver cell line and HCC cell lines can produce IL-6 so that Kupffer cells are noit the only source of the cytokine in the liver well as other immune cells. That the fact that HCC cells reacted to stimulation of biological molecules such as liposome, dsRNA or cell debris with increasing production of IL-6 indicates that the cytokine might play an important role not only in the period of tumor initiation but progression and recurrence as well.

Expression of Oct4 in HCC and modulation to wnt/ß-catenin and TGF-ß signal pathways.

Hepatocellular carcinoma (HCC) is considered as a disease of dysfunction of the stem cells. Studies on stem cells have demonstrated that Oct4 plays a pivotal role in embryo regulation. In order to understand the role of Oct4 in HCC and the relationship among Oct4 and wnt/ß-catenin and TGF-ß signal pathways, we have detected the expression of Oct4, Nanog, Sox2, STAT3 as well as the genes in wnt/ß-catenin, and TGF-ß families in HCC cell lines and in tumor specimens from HCC patients. The authors found that Oct4 was expressed in all of the four HCC cell lines and the tumor specimens from HCC patients. Some other genes were also expressed in them with different level including Nanog, Sox2, STAT3 and TCF3, wnt10b, ß-catenin, ELF, Smad3 and Smad4. The ability of the clone formation and migration of the HepG2 decreased after Oct4 was knockdowned. Silencing of Oct4 and TCF3 in HCC cell line HepG2 revealed that there were complicated relationships among Oct4, wnt/ß-catenin family and TGF-ß family genes. Knockdowning Oct4 reduced the expression of TGF-ß family genes ELF, Smad3, Smad4 and wnt/ß-catenin family genes, wnt10b, and ß-catenin but increased TCF3. In reverse, knockdowning TCF3 led to the increased expression of Oct4 and TGF-ß family genes. In conclusion, the expression of Oct4 in HCC may play an important role as in stem cell. Because Oct4 improves not only the function of wnt/ß-catenin, but also the TGF-ß signal pathways, the significance of its expression in HCC might be more complicated than we evinced before.

[Analysis on the expression of the stem cells related genes in hepatocellular carcinoma cell lines].

OBJECTIVE: To investigate the expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines of SMMC-7721, Bel-7402, HepG2, MHCC-97 and normal hepatocellular cell line of L02, and to compare the response of these cell lines to all-trans retinoic acid. METHODS: RT-PCR was used to detect expression of Oct4, Sox2, Nanog, SMO, beta-Catenin and Wnt5b mRNA in four hepatocellular carcinoma cell lines and normal hepatocellular cell line. Real time-PCR was used to quantify the expression of the genes. RESULTS: There are different levels of expression of the stem cell-related gene in hepatocellular carcinoma cell lines and control cell line (P less than 0.05). There are significant differences in HepG2 and L-02 for the response to all-trans retinoic acid (P less than 0.05). CONCLUSIONS: The stem cell-related genes are differentially expressed in different hepatocellular carcinoma cell lines.

Anticancer drugs are synergistic with freezing in induction of apoptosis in HCC cells.

Cryotherapy has been shown to be an important therapeutic alternative to surgery in the treatment of hepatocellular carcinoma (HCC). Here, the influence of cryo-chemotherapy on HCC was examined in vitro using the human HCC cell line Bel-7402, a drug-resistant HCC cell line originating from Bel-7402 cells (Bel-7402/R), as well as two control cell lines, the HCC cell line SMMC-7721 and a colorectal tumor cell line HIC-251. Cells were treated with either exposure to different freezing temperatures (ranging from -15 to -80 degrees C for 20 min), exposure to sub-lethal concentrations of anticancer chemotherapy drugs or a combination of cryotherapy and chemotherapy. Cell viability and apoptosis under each condition were investigated. We found that the combined treatment resulted in increases in both cell death and apoptosis compared to either treatment alone. The increased level of apoptosis observed in Bel-7402 cells after cryo-chemotherapy was inhibited in the presence of caspase inhibitors. Furthermore, Bax expression was increased 2- to 3-fold in cells exposed to the combination treatment compared with cells treated by freezing or drugs alone. In contrast, Bcl-2 levels remained constant. Although Bel-7402/R cells originated from the Bel-7402 cell line, they were more sensitive to the freezing procedure than the parental cell line. The level of Bax expression in Bel-7402/R cells was also higher than that observed in the parental cell line. In addition, we found that Bel-7402/R cells had lower levels of survivin mRNA than the parental Bel-7402 cells, in both untreated and treated cells. In conclusion, our data show that in HCC cells, apoptosis induced by cryotherapy can be synergistically enhanced using anticancer drugs.

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402/R.

AIM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2,000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhIFN-gamma, monoclonal anti-CD3 antibody, rhIL-1alpha as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5 x 10(10) of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.